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Nir Barzilai M.D. Director: Institute for Aging Research Departments of Medicine and Molecular Genetics Cambridge, September 2003 Thank you longevity!!! Longevity-biology of caloric restriction Longevity-genes Xiao-Hui Ma M.D Xiao-man Yang M.D. Radhika Muzumdar M.D. Bing-Qian Liu M.D Ilan Gabriely M.D. Patricia Vuguin M.D. Gil Atzmon Ph.D. Program project Jill Crandall M.D. Meredith Hawkins M.D. Harry Shamoon M.D. Luciano Rossetti M.D. Michael Brownlee M.D. Silvana Obici M.D. Bill Greiner R.N. Magda Gabriely M.D. Debra Davidson M.Sc. Clyde Schechter M.D. Richard Lipton M.D. Ms. Marion Maze Ms. Linda Radner Other Collaborators Alan Shuldiner M.D. (UMD) Braxton Mitchell Ph. D. (UMD) Hassy Cohen M.D. (UCLA) Tom Perls M.D. (BU) Gad Rennert M.D., Mp.H.(Technion) Amiela Globerson Ph.D. (Ben Gurion) Norman Fleischer M.D (Einstein) Philip Scherer Ph.D. (Einstein) Becky Simmons M.D. (Penn) 65 millions year 50 years Antagonistic Pleiotropy 65 millions year 50 years The metabolic syndrome of aging: Insulin resistance High insulin Obesity/abdominal obesity Type 2 diabetes mellitus Dyslipidemia Low HDL, ‘small’ LDL Hypertension AGT Inflammation IL-1, IL-6, CRP Endothelial dysfunction Thrombosis PAI-1 ASCVD Cancer How do nutrients induce biological effects? Nutritional (transcription) regulation of peptides Leptin 2 am Glucose-insulin/FFA/AA 8 am Barzilai JCI 1997, Rossetti & Barzilai Nature 1999, Gabriely & Barzilai Diabetes 2002, Xma & Barzilai J Gerontol 2002. Leptin fails to regulate body fat distribution, insulin action, and endocrine functions with aging 8 am How is leptin expression regulated? Hexosamine Biosynthetic Pathway Glucose AA (Glutamine) Glucosamine Glc-1-P Glucose-6-P GFAT 1% UDP- Glc --3-6% GlcN-6-P F -6-P FFA Triose -P UDPGlcNAc Glycogen Glycolysis Glycosylation Sp1 NFkB PGC1 FFA How can a pathway serve as ‘nutrient sensing’? • Should serve as a switch “on” or “off”. • Should be a small pathway that can be amplified by several folds. • Should be linked to a biological effect (transcription). • Should exist in every organ, although may be distinct. • Should apply to all models of CR Is the HPB activated with aging? Muscle UDPGlcNAc/UDPGlc ** * 3 2 1 0 Young Old AL Old CR Although old CR animals are metabolically similar to young rats they have increased levels of muscle hexosamines (p<0.01) Does the HBP effect the muscle function of old CR rats? 3 mo (n=5) and 21 mo (n=5) CR S-D rats Insulin-3 mU/kg/min GlcN-7 µM/kg/min/or Saline Rd (mg/kg/min) 30 **** 25 Young/old CR saline control **** UDP-GlcNAc 20.8 nmol/g muscle 20 **** Young Old 15 10 5 P<0.001 UDP-GlcNAc 17.7 nmol/g muscle 290 210 230 250 270 90 110 150 190 30 50 70 0 Time (min.) Excess nutrients can induce peripheral insulin resistance through the HBP! (and more so in aging) Does the HBP affect adipose tissue function of old CR rats? PAI-1 expression (Real-time PCR) 14 12.5 12 10 8 Young Old 6 4 3.2 2.2 2 1 0 Saline GlcN Variety of (harmful?) fat-derived peptides are over expressed through the HBP. Is the transcription of PAI-1 by the HBP associated with increase in plasma levels? PAI-1 activity U/ml 30 * 25 20 15 * * 10 5 0 Glc + - Young Artheriosclerosis. 160: 117. 2002. + - Old CR ß-actin PAI-1 30 (relative to baseline) Gene expression level Is increased availability of FFA effect PAI-1 transcription in humans adipose tissue? 20 10 0 Liposyn - + Hawkins et al Similar effects have been induced by hyperglycemia, hyperinsulinemia, and increased levels of amino acids in humans and rodents Which organ is most important to exert the biological effects of nutrient excess on longevity? Does VF account for the effects of caloric restriction on insulin action in aging rats? Young Old SC- Old VF- Old CR 500 400 300 200 100 0 Body Weight (g) Diabetes; 51:2951, 2002 Is the removal of VF sufficient to restore insulin action? Young SC- VF- CR 30 * 20 10 0 Rd (mg/kg LBM/min) *P<0.01 Diabetes; 51:2951,2002 Does ‘knock out’ of VF prevents diabetes in Zucker diabetic rats? %DM 120 ~80% 100 80 ZDVFZDVF+ 60 40 ~40% 20 0 2 mo 3 mo Diabetes; 51:2951,2002 4 mo 5 mo 6 mo PAI-1 16 14 12 10 8 6 4 2 0 $$ 12 10 8 * Gene expression (adjusted by Gene expression (adjusted by GAPDH) GAPDH) Gene expression (adjusted by GAPDH) $$@ Saline Glucose Insulin GlcN+In $$ @ Saline Glucose Insulin GlcN+In 20 $ 0 $$ 10 0 TNF- 5 4 3 2 1 0 $$$ 30 4 6 Resistin $$$ $ 40 6 2 60 50 $$ Angiotensinogen 20 18 16 14 12 10 8 6 4 2 0 @ Saline Glucose Insulin GlcN+In Gene expression (adjusted by GAPDH) $@ Saline Glucose Insulin GlcN+In Leptin Saline Glucose Insulin GlcN+In $@@ $$ $$ Acrp30 Gene expression (adjusted by GAPDH) 50 45 40 35 30 25 Gene expression (adjusted by GAPDH) Gene expression (adjusted by GAPDH) Nutrients, nutrient sensing, and induction of fat-derived peptides 16 14 12 10 8 6 4 2 0 Saline Glucose Insulin GlcN+In @ $$ $$ How do nutrients induce biological effects? FAT+Nutritional (transcription) regulation of peptides Leptin 2 am PAI-1 AGT IL-6 IL-1 Resistin SAA 8 am Glucose/insulin/FFA Clinical trials are standardizes by fasting levels, and are Under estimating daily transcription of peptides! 8 am You always understand the results of your experiment! But You never know the whole truth! 100 98 92 95 Lipoprotein particle sizes with age 9.8 21.6 9.6 21.4 LDL Particle Size (nm) HDL Particle Size (nm) Heritabilty of lipoprotein size 0.4-0.7! 9.4 9.2 9.0 21.2 21.0 Probands 20.8 8.8 20.6 8.6 20.4 60 65 70 75 80 Age 85 90 95 100< Control Offspring 60 65 70 75 80 85 90 95 100< Age JAMA (October 03 ) Is lipoprotein sizes associated with HTN or CVD? (in offspring of centenarians) 60 * 70 * 60 50 Healthy (n=209 HTN (n=64) CVD (n=20) 50 40 40 30 30 20 20 % large HDL size *P<0.003 % large LDL size JAMA (October 03 ) Cholestryl Ester Transfer Protein (CETP) Exon 14 TESSSESIQSFLQSMIT Homozygous II = Isoleucine GTC = Valine A A A ATC OR G Heterozygous IV G OR G Homozygous VV CETP I 405 V frequency in families with exceptional longevity Control Offspring 70 Percentage in population Probands 60 50 40 30 * 20 *** 10 0 II IV VV CETP VV genotype attributes to 18% of longevity JAMA (October 03 ) IS CETP genotype associated with its plasma levels? 2.4 I/I I/V V/V 2.2 2 1.8 * 1.6 1.4 1.2 1 CETP levels (ug/ml) *P<0.02 JAMA (October 2003 ) Is lipoprotein sizes a risk for MS ? (in offspring and spouse of offspring) 70 *** 65 60 *** Healthy MS (n=47) (n=221) 55 60 50 55 50 45 45 40 40 35 35 30 30 % large HDL size ***P<0.001 % large LDL size JAMA (2003, 290;15;2030-40 ) APOC3 C(-641)A frequency in families with exceptional longevity Control Offspring 70 Probands Percentage in population 60 50 40 30 20 *** *** 10 0 CC ***P<0.001 CA AA Rate limiting steps for humans longevity 100 80 60 Alive cancer 40 CVD CR 20 Telomeres Mitochondria 0 60 80 100 120 140 Age Barzilai lab Revisiting the role of fat mass in the life extension induced by caloric restriction • J. Gerontol Barzilai & Gupta; 1999, 54:B89 The role of fat mass as causative of age-related diseases leading to morbidity and mortality is well accepted. Type 2 DM is a striking example. ...This is not a novel hypothesis Mounting evidence supports the notion that fat mass is inert and plays no role, as compared with nutrients per se in CR. …This hypothesis should not be revisited Rueben Andres Fault! * @ $@ $$ $$ SC Leptin 12 10 8 $$ * $$ * @ $$@ $$ 0 VF $$ 10 SC VF VF TNF- 6 5 $$$ 0 Acrp30 Saline Glucose Insulin GlcN+In 4 * 3 2 $@@ $$ $$ 1 0 SC $ 20 SC Gene expression (adjusted by Gene expression (adjusted by GAPDH) GAPDH) Gene expression (adjusted by GAPDH) Saline Glucose Insulin GlcN+In $$$ 30 @ $ Angiotensinogen 20 18 16 14 12 10 8 6 4 2 0 60 50 2 VF Resistin Saline Glucose Insulin GlcN+In 40 6 4 @ Saline Glucose Insulin GlcN+In Gene expression (adjusted by GAPDH) 16 14 12 10 8 6 4 2 0 Saline Glucose Insulin GlcN+In SC VF Gene expression (adjusted by GAPDH) PAI-1 50 45 40 35 30 25 Gene expression (adjusted by GAPDH) Gene expression (adjusted by GAPDH) Nutrients, nutrient sensing, and induction of fat-derived peptides 16 14 12 10 8 6 4 2 0 Saline Glucose Insulin GlcN+In @ $$ $@ $ $ SC VF Probability of survival in caloric restricted animals 100% Survival Decrease in all causes of deaths Months Human longevity genes We hypothesize that to get to age 100 (1/10,000) one needs longevity assurance genes. Offspring inherit longevity traits from their parents, and these may be a platform for longevityassociated traits. We have collected over 1000 samples! (At their homes) Our population of (Ashkenazi) Jews is genetically homogenous, increasing the likelihood of genetic discoveries. Funding by AECOM the National Institute of Aging and the Ellison medical Foundation Lipoprotein sizes and Cognitive function (by MMSE) in centenarians 100 21.5 80 21.2 60 20.9 40 20.6 20 20.3 0 20 % large LDL size MMSE>25 MMSE<25 LDL size (nm) Lipoprotein sizes and I405V genotype 70 60 * * 60 50 I/I I/V V/V 50 40 40 30 30 20 20 % large HDL size *P<0.001 vs. I/I % large LDL size JAMA (In Press ) Does leptin resistance effect insulin action with aging? % suppression of hepatic glucose production Leptin vs. pair-fed 0 -20 * -40 -60 -80 -100 Young Gabriely et al Diabetes 51:1016, 2002 Old CR Leptin resistance may lead to alteration in body fat-distribution and insulin resistance! Mitochondrial Overproduction of Superoxide Activates Four Major Pathways of Hyperglycemic Damage By Inhibiting GAPDH NADP + NADPH Glucose NAD + NADH Sorbitol Fructose Polyol pathway Glucose-6-P GFAT Glucosamine-6-P Fructose-6-P Gln UDP-GlcNAc Glu Hexosamine pathway NADH DHAP NAD + -Glycerol-P DAG PKC Protein kinase C pathway Glyceraldehyde-3-P NAD + GAPDH NADH O2– • Methylglyoxal AGEs AGE pathway 1,3-Diphosphoglycerate Brownlee M. Nature. 2001;414:813-820. Does the HBP affect adipose tissue function of old CR rats? Leptin expression (Real-time PCR) 12 11 10 8 Young Old 6 4 3.2 2.3 2 1 0 Saline GlcN Does leptin fail to regulate VF with aging? % decrease Leptin vs. pair-fed 0 -10 -20 * -30 -40 -50 -60 Young Barzilai JCI 1997, Rossetti & Barzilai Nature 1999 Gabriely & Barzilai Diabetes 2002, Xma & Barzilai J Gerontol 2002. Old CR Leptin resistance may lead to alteration in body fat-distribution and to insulin resistance! Out off approximately 7,000 full-length sequences and approximately 1,000 EST clusters 2570 were expressed 200 were up/down- regulated in each chip Atzmon et al Horm Metab Res. 2002 34:622 Gene Insulin-induced growth-respons protein (CL-6) Beta 3-adrenergic receptor Phosphoenolpyruvate carboxykinase (GTP) PEPCK PPAR-gamma Hormone sensitive lipase Insulin-like growth factor I Fatty acid transporter Thioesterase II Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase Low molecular weight fatty acid binding protein GLUT1 = glucose transporter 1 Lipopolysaccharide binding protein Lysosomal acid lipase = intracellular hydrolase Fatty acid synthase Type II cAMP-dependent protein kinase regulatory subunit Adipocyte hormone-sensitive cyclic AMP phosphodiesterase Potential-sensitive polyspecific organic cation transporter Retinol-binding protein (RBP) gene, exon 5 Steroidogenic acute regulatory protein Growth hormone receptor Chaperonin 60 (Hsp60) and chaperonin 10 (CPN10) genes, nuclear genes encoding mitochondrial proteins Aquaporin 7 Angiotensinogen Glutathione-dependent dehydroascorbate reductase MHC class II antigen RT1.B-1 beta-chain Tricarboxylate carrier Thyroid stimulating hormone receptor Phosphodiesterase I Water channel aquaporin 3 (AQP3) Carbonic anhydrase II Wistar-Kyoto (Heidelberg) angiotensin converting enzyme GST Na-K-Cl cotransporter (Nkcc1) Alpha-2-u globulin Glutathione S-transferase Yc1 subunit Wistar transforming growth factor beta-3 Liver glutathione S-transferase Yc subunit Polymeric immunoglobulin receptor Alkaline phosphatase Multiple increase In VF In SC 5.9 5 4.3 4.1 3.5 3.2 3.2 156 21.1 10.1 9.8 9.7 4.6 3.9 7.4 5.7 5.6 5.2 5 5 4.8 - 4.3 4.5 4 3.6 3.6 3.6 3.5 - 20.8 11.8 7.4 6.1 5.7 5.3 4.7 4.7 4.4 4 3.9 Pathways Glucose homeostasis Insulin action and lipid metabolism Cellular metabolism and other Atzmon et al Horm Metab Res. 2002 34:622